The proposed project seeks to overcome a major barrier in the study of mutations in evolution by experimentally determining the distribution of fitness effects of every nearest neighbor variant (variants that differ from the wildtype sequence by a single base pair mutation) of the gene encoding beta- lactamase inhibitory protein (BLIP). Specifically, this research aims to create a library of every nearest neighbor variant of BLIP on the DNA and on the amino acid level and to quantify the fitness of these variants. Library creation will be accomplished using Kunkle mutagenesis, a site-directed mutagenesis method that can produce a high percentage of DNA with the desired mutations. Fitness will be characterized based on the ability of BLIP variants to inhibit TEM-1 beta-lactamase (an antibiotic resistance enzyme that hydrolyzes beta-lacam antibiotics, such as ampicillin). E. coli will be transformed with the BLIP library and a unique band-pass selection system will be utilized to separate the library into bins based on the minimal inhibitory concentration (MIC) for ampicillin. Because the minimum inhibitory concentration inversely correlates to the function of BLIP, the library will be sorted into sub-libraries that reflect relative fitness. High-throughput sequencing technology will be used to determine the DNA sequence of every library member. In this way, a complete fitness distribution due to single base pair mutations for the entire gene wil be determined. This result will address a number of questions about mutations, including the contributions of synonymous versus non-synonymous mutations, and how fitness effects are distributed in the linear sequence and in the three-dimensional structure of BLIP. This knowledge of mutational effects has the potential to significantly improve understanding in the fields of evolutionary biology, protein engineering, and medical genomics. The outcomes of this research will also be of medical significance owing to the increased understanding of an antibiotic resistance inhibitor and the possibility of finding an improved BLIP variant. PUBLIC HEALTH RELEVANCE: This project focuses on the study of mutations, which are the cause of genetic diseases and disorders. Understanding the global picture of how these mutations affect proteins would be beneficial to the prevention and treatment of these diseases and disorders. Furthermore, because the model gene for this research encodes a protein that inhibits antibiotic resistance, the proposed project will also be beneficial in developing improved antibiotic resistance inhibitors for the treatment of bacterial infection.